Several important physiological processes in the cornea have recently been demonstrated to be affected by agents which increase cyclic AMP in cells. These effects include corneal transparency, epithelial cell mitosis and release of collagenase. We propose an investigation of the underlying biochemical mechanisms involved in the mediation of the demonstrated effects of cyclic AMP on corneal epithelium and other corneal layers. The characteristics of cyclic AMP-dependent protein kinase(s) will be studied in both the cytosol and particulate fractions of bovine and human corneal epithelial cells. Employment of a recently developed microtechnique will not only allow the detection of these activities in small quantities of tissue, but also provide other valuable information of the regulation of cyclic AMP-dependent protein kinase(s) in the cornea. Characterization of the hormone-sensitive adenylate cyclase will be performed on partially purified bovine corneal epithelial membranes. These studies will indicate which hormones and drugs are capable of directly stimulating (or inhibiting) synthesis of cyclic AMP in corneal epithelium. Information provided by the above studies on broken cell preparations will then be used to develop protocols for perfusion of bovine and human corneas to investigate correlations between the following: drug or hormone administration; cyclic AMP concentration; protein kinase activity; phosphorylation of cellular proteins by protein kinase; and observance of ultimate physiological responses in the cornea. Identification of the substrates phosphorylated by corneal cyclic AMP-dependent protein kinase(s) and an appreciation of their respective roles in development of the physiological responses is the ultimate goal of the proposed research. This knowledge whould provide the basis for the development of pharmacological treatments of the many corneal disorders which involve enzymes regulated by cyclic AMP.